CN115011711B - Kit and method for visually detecting Edwardsiella ictaluri of zebra fish source - Google Patents
Kit and method for visually detecting Edwardsiella ictaluri of zebra fish source Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting Edwardsiella ictaluri of zebra fish source in a closed tube visualization way, wherein a primer is designed according to a hypothetical protein epi18 in a conserved protein of Edwardsiella ictaluri, and the aim of judging a target gene in the closed tube visualization way can be fulfilled through a pH sensitive indicator in a reaction system; and the method can also directly extract DNA from the brain tissue of the diseased fish to carry out rapid detection, has high speed and high visualization degree compared with the original detection method, and has good field and resource-limited basic laboratory detection application prospects.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a kit and a method for visually detecting Edwardsiella ictaluri which is a zebra fish source.
Background
Zebra fish is the experimental fish which is internationally applied to the most widely studied science, and the health condition and the quality of the fish are the basis for developing all research works related to the zebra fish. In the indoor breeding process of zebra fish, the development of related research work based on zebra fish is severely restricted by the outbreak of various bacterial diseases. Edwardsiella ictaluri (Edwardsiella ictaluri) is reported to be a bacterial pathogen with serious hazard in zebra fish houses, once the bacterial pathogen bursts in zebra fish laboratories, the bacterial pathogen spreads rapidly, the death rate is as high as 50%, the loss is huge, the zebra fish which is infected and survived can still carry the pathogen, and the feces of the zebra fish can be swallowed by healthy fish, or the bacterial pathogen can be transmitted to other healthy fish through ingestion of infected fish carcasses to cause a second outbreak of the fish disease. The diseased fish often shows symptoms such as swelling of the fish belly, obvious congestion and bleeding of the parts such as the oral cavity, the mandible, the eye orbit, the gill cap, the anus, the basal part of fin, etc., subcutaneous redness in the middle part of the head top, craniofacial skin necrosis and ulcer on the back of the head, and severe skull cleavage and brain tissue exposure.
At present, the detection method of the Edwardsiella ictaluri comprises conventional separation culture (at least 2 days are required for separation culture), physiological and biochemical identification, polymerase chain reaction, 16S rDNA sequencing and the like. The conventional method has the defects of long detection time, time consumption, labor consumption and the like; the enzyme-linked immunosorbent assay (ELISA) can rapidly detect bacteria, but related antibodies are required to be prepared first, and the steps are tedious and time-consuming. The PCR technology has the advantages of sensitivity, specificity, rapidness and the like, and is widely applied to the detection of the aquaculture pathogen. However, the PCR technology has high requirements on laboratory personnel and environment, and the equipment are expensive and the operation steps are more. Compared with the common PCR technology, the fluorescent PCR detection technology simplifies the operation steps, can eliminate the cross contamination caused by amplified products and reduces the occurrence of false positives. However, real-time fluorescent PCR equipment is expensive and cannot be used for on-site detection. Therefore, a novel method for rapidly, accurately and sensitively detecting Edwardsiella ictaluri is established, and has important significance for early diagnosis, early warning, real-time monitoring and timely treatment of diseases.
Loop-mediated isothermal nucleic acid amplification (LAMP) is a novel nucleic acid amplification technique invented by Notomi T et al (PMID: 10871386). The technology relies on 4 specially designed primers and DNA polymerase with strand displacement property, and the reaction is completed under the constant temperature condition of 61-65 ℃, so that multiple temperature change processes such as denaturation, annealing, extension and the like of PCR amplification are avoided, and the production cost of reaction equipment is greatly reduced; meanwhile, the method can still keep the advantages of high sensitivity, specificity and the like equivalent to the PCR technology, and shows huge activity in the field of rapid detection. In recent years, the technology has been widely used for pathogen detection at home and abroad.
At present, in Edwardsiella ictaluri detection of other species isolation, there has been reported a LAMP technique (PMID: 16157211) in foreign Yeh et al. However, the practical detection shows that the primer used in the report does not work in the detection of the Edwardsiella ictaluri, and the LAMP result is judged and mainly realized by agarose electrophoresis, the LAMP amplification product yield is huge, and the pollution problems such as aerosol and the like are very easy to generate in the uncapping detection process, so that false positive results appear. In 2015, studies have reported that a large amount of H is generated during the nucleic acid isothermal amplification reaction accompanied by accumulation of the amplified product of the target gene + The pH of the reaction system was lowered (PMID: 25652028). Therefore, the purpose of judging the closed tube visual result can be achieved by judging whether the LAMP reaction occurs or not through color change by utilizing the pH sensitive indicator. At present, no related report of the application of a pH indicator to LAMP detection of Edwardsiella ictaluri of zebra fish source is known.
Disclosure of Invention
The invention provides a kit and a method for detecting Edwardsiella ictaluri of zebra fish source in a closed tube visualization way, wherein a primer is designed and designed according to a hypothetical protein epi18 in a conserved protein of Edwardsiella ictaluri, and a pH indicator is added into a reaction system, so that the purpose of judging a target gene in the closed tube visualization way can be realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
LAMP primer combination for detecting Edwardsiella ictaluri of zebra fish source: the outer primer sequences are shown in SEQ ID NO.1 and 2, and the inner primer sequences are shown in SEQ ID NO.3 and 4.
The kit for detecting the Edwardsiella ictaluri comprises outer primers with sequences shown as SEQ ID NO.1 and 2 and inner primers with sequences shown as SEQ ID NO.3 and 4.
Preferably, the concentration ratio of the outer primer to the inner primer is 1:4-8.
Preferably, the kit further comprises LAMP reaction premix (containing a pH indicator), and the positive plasmid is a plasmid containing Edwardsiella ictaluri epi18 gene.
Compared with the prior art, the invention has the following technical effects:
1. the LAMP detection method for the Edwardsiella ictaluri of zebra fish sources is high in specificity, the specific primers are designed according to the hypothetical protein epi18 in the Edwardsiella ictaluri conserved protein reported in the literature, and a pH indicator is added into a reaction system, so that the effect of visually judging whether a target gene is effectively amplified by closed tubes is achieved, and the false positive problem caused by methods such as agarose gel electrophoresis and fluorescent dye can be effectively avoided.
2. The invention has high detection sensitivity, and the lowest detection rate of the genome DNA is 7.9 copies/. Mu.L; the detection time is short, and the amplification reaction only needs 60 minutes; the Edwardsiella ictaluri pathogen can be directly detected from the brain tissue DNA of the diseased fish, the whole detection process only needs 70 minutes from the extraction of the sample genome DNA to the judgment of the completion result, compared with the conventional method for separating and culturing the pathogen, the pathogen genome DNA is extracted, and then the pathogen is confirmed by a PCR (polymerase chain reaction) equivalent method or a physiological biochemical method, so that the detection speed is greatly improved; the method has the advantages that the requirements on instrument and equipment are low, a PCR instrument, a gel electrophoresis and imaging system and the like used for conventional PCR are not needed, the detection can be completed only by one water bath, the operation is simple, the result is obvious, the whole detection process does not involve complex instruments and equipment, the operation can be completed by personnel with a little molecular biology basis, the detection result is clear and obvious, the judgment can be realized by naked eye observation, and the method has good field and limited-resource basic laboratory detection application prospects.
Drawings
FIG. 1 shows the results of the sample test in example 1. 1 is a sample to be detected, 2 is a positive control group, and 3 is a negative control group.
FIG. 2 shows the sensitivity test results in example 2. 1-6 are respectively 10-fold diluted genome templates, and the copy number of the epi18 of the target gene in the corresponding templates is 7.9X10 5 、7.9×10 4 、7.9×10 3 、7.9×10 2 、7.9×10 1 、7.9×10 0 (copy/. Mu.L), 7 is a negative control.
FIG. 3 shows the results of detection of Edwardsiella ictaluri by the primer electrophoresis method for detecting Edwardsiella ictaluri provided in the literature in embodiment 3. 1 is DL2000 Marker,2 is Edwardsiella ictaluri, and 3 is negative control.
FIG. 4 shows the results of the visual detection of Edwardsiella ictaluri by using the primers for Edwardsiella ictaluri in example 3. 1 is Edwardsiella ictaluri, and 2 is negative control.
FIG. 5 shows the results of the specific assay in example 3. 1 to 6 are Edwardsiella ictaluri, shigella, vibrio, aeromonas hydrophila, aeromonas caviae, escherichia coli, and 7 is a negative control.
FIG. 6 shows the LAMP detection result of a pathogen in the brain tissue of a zebra fish disease in example 4. 1-4 are respectively a pathogenic brain tissue DNA template, a positive Edwardsiella ictaluri DNA template, a healthy fish brain tissue DNA template and a sterile water template.
Detailed Description
Example 1: establishment of method for detecting Edwardsiella ictaluri by LAMP technology
(1) LAMP primer design: according to the reported conserved protein epi18 gene of Edwardsiella ictaluri, the coding sequence of the gene is searched in NCBI, the outer primers are designed to be F3a and B3a, the plasmid of the Edwardsiella ictaluri which is separated and preserved in the national zebra fish resource center is used as a template, PCR amplification is carried out, the base sequence of the sequence is confirmed by sequencing the amplified product, the inner primers of LAMP are designed to be FIPa and BIPa on the basis, and the primer sequence is shown in table 1.
TABLE 1 LAMP detection primer sequences of Edwardsiella ictaluri
(2) Extracting genome DNA of pathogenic bacteria: edwardsiella ictaluri strain (E.ictaluri) E1 (Liu Liyue, etc.), which is independently separated from the zebra fish resource center in the-70 ℃ long-term preservation state, is used for separating and identifying the pathogen of the zebra fish 'split head disease' and diagnosing common diseases, and 2022) is streaked on a BHIA solid culture medium, and is cultured overnight at 30 ℃. Picking monoclone into 5mL BHI liquid culture medium, shake culturing at 30deg.C at 165r/min for about 10 hr, extracting genome DNA from the cultured thallus overnight with commercial kit (Tiangen Biochemical Co., ltd.), and storing at-20deg.C. Or centrifuging the cultured thallus overnight, adding into 0.5mL TE buffer (pH 8.0) or double distilled water, shaking, mixing, boiling at 95-100deg.C for 3-8 min, centrifuging at 10,000r/min to obtain supernatant, and storing at-20deg.C.
(3) Preparation of positive control: PCR amplification is carried out by taking genome DNA of Edwardsiella ictaluri strain E1 as a template and F3 and B3 of epi18 target genes as primers. PCR reaction System (50. Mu.L): 2X Rapid Taq Master Mix (Vazyme) 25. Mu.L, 2. Mu.L of each primer, 1.0. Mu.L of template, and 20. Mu.L of ultrapure water. The reaction procedure: 94 ℃ for 5min;94 ℃ for 30s,55 ℃ for 30s and 72 ℃ for 1min, and 30 cycles are total; and at 72℃for 10min. The product is subjected to 2% agarose gel electrophoresis, is connected to a pMD-18T vector after gel cutting recovery, converts competent cells, and is subjected to sequencing verification, and positive plasmid pMD-18T-epi18 is extracted as a positive control and is preserved at-20 ℃ for later use.
(4) Negative control preparation: ultrapure water.
(5) Sample detection and result judgment: 1 μl of genomic DNA is added into a LAMP reaction system, wherein the reaction system (25 μl) contains LAMP mix 12.5 μl (containing pH indicator), 1 μl of each of outer primers 10mM F3a and 10mM B3a, 4 μl of each of inner primers 10mM FIPa and 10mM BIPa, 1.5 μl of ultrapure water, 1 μl of template, and pH of the 1M KOH adjustment system is 7.5-9.5. After heating at 65℃for 60min and 85℃for 3min, the results were judged by: the color of the reaction system is observed by naked eyes, if the color of the epi18-LAMP reaction system is similar to that of a positive control, the reaction system turns yellow, and the reaction system is positive, so that the sample to be detected contains Edwardsiella ictaluri; if the color of the epi18-LAMP reaction system is similar to that of the negative control, namely, the original color of the reaction system is kept to be pink, the reaction is negative, which indicates that Edwardsiella ictaluri is not contained in the sample to be detected (figure 1).
Example 2: sensitivity of closed tube visualization loop-mediated isothermal amplification technology of zebra fish-derived Edwardsiella ictaluri
The pMD-18T-epi18 positive plasmid constructed in example 1 was used as a template, 10-fold dilution was performed, and the number of copies of epi18 of the target gene in the template was 7.9X10, respectively 5 、7.9×10 4 、7.9×10 3 、7.9×10 2 、7.9×10 1 、7.9×10 0 (copy/. Mu.L) the LAMP reaction was carried out using sterile deionized water as a negative control, and the LAMP reaction system and procedure described in example 1 was used, with the reaction system being 25. Mu.L. The results of the samples tested were judged by visual inspection of the changes in example 1. As shown in FIG. 2, the detection result that the copy number of the target gene epi18 is greater than or equal to 7.9 copy/. Mu.L is positive, and the result of the negative control is negative, which shows that the sensitivity of the method for amplifying the Ictalurus ictaluri assumed gene epi18 is 7.9 copy/. Mu.L.
Example 3: specificity of closed tube visualization loop-mediated isothermal amplification technology of zebra fish-derived Edwardsiella ictaluri
Regarding the detection of Edwardsiella ictaluri separated from other species, there has been a report (PMID: 16157211) on LAMP technology by foreign Yeh et al, and the results of detecting Edwardsiella ictaluri of zebra fish origin by using the primer and electrophoresis detection method provided in the document are shown in FIG. 3, but the detection results require electrophoresis confirmation, and the defects of complicated operation, easy generation of aerosol pollution and the like are present; if the detection primer provided by the document is used for detecting Edwardsiella ictaluri by using the closed tube visual detection method, the detection result is shown in fig. 4, and the Edwardsiella ictaluri can not be successfully detected by using the LAMP technology published by Yeh et al abroad, the Edwardsiella ictaluri detection method is not feasible for detecting Edwardsiella ictaluri by using the closed tube visual detection method, the method is only suitable for detecting Edwardsiella ictaluri by electrophoresis, electrophoresis and imaging equipment are arranged at a detection place, toxicity still exists in the preparation of electrophoresis gel and gel running process, and compared with the visual detection, the method has no advantage.
In order to detect the specificity of the kit, the LAMP detection method in example 1 is adopted, shigella, vibrio, aeromonas hydrophila, aeromonas guinea, and escherichia coli are respectively used as templates, and negative control with sterile deionized water is set as the templates to carry out a specificity test. The detection results are shown in fig. 5: edwardsiella ictaluri is specifically amplified, and shigella, vibrio, aeromonas hydrophila, aeromonas guinea, escherichia coli and negative control are not amplified. The results show that the LAMP detection method disclosed by the invention can specifically and successfully amplify the target sequence of the Edwardsiella ictaluri without cross reaction with other bacterial nucleic acid, and has good specificity and no false positive.
Example 4: closed tube visualized loop-mediated isothermal amplification technology for detecting Edwardsiella ictaluri in zebra fish disease fish
DNA was extracted from top-of-head perforated, transswimming zebra fish: taking 10mg to 1.5mL centrifuge tube of diseased fish brain tissue, rapidly extracting tissue genome DNA by using a commercial kit (Novain company), and storing at-20 ℃ for later use. LAMP detection of Edwardsiella ictaluri was carried out by using the LAMP reaction system and procedure described in example 1, and the reaction system was 25. Mu.L. The Edwardsiella ictaluri DNA template is used as a positive control, the asymptomatic healthy zebra fish brain tissue DNA template is used as a negative control, and the sterile water template is used as a blank control, and the result is shown in figure 6, and the group of the ill fish and the positive control group are yellow, and the other groups are pink, so that the presence of Edwardsiella ictaluri infection of the ill fish is judged.
Sequence listing
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<120> closed tube visual detection kit and method for Edwardsiella ictaluri of zebra fish source
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Claims (3)
1. The kit for detecting the Edwardsiella ictaluri is characterized by comprising an LAMP primer combination and a pH indicator, wherein the outer primer sequence of the LAMP primer combination is shown as SEQ ID NO.1 and SEQ ID NO. 2, and the inner primer sequence is shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. The kit of claim 1, wherein the concentration ratio of the outer primer to the inner primer is 1:4-8.
3. The kit of claim 1 or 2, further comprising a LAMP reaction premix, a positive plasmid, wherein the positive plasmid is a plasmid containing the edwardsiella ictaluri epi18 gene of zebra fish source.
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